Process for preparing ryanodine



Patented Aug. 14, 1951 PROCESS FOR PREPARING RYAN ODINE Edward F. Rogers, Middletown, N. J., assignor to Merck & 00., Inc., Rahway, N. J a corporation of New Jersey No Drawing. Application September 18, 1947, Serial No. 774,895

4 Claims. (Cl. 260--236) This invention relates to chemical compounds having insecticidal activity, and more particularly to the new insecticidal alkaloid, ryanodine, and to the process for preparing the same in pure crystalline form.

In United States Patent No. 2,400,295, issued May 14, 1946, to Folkers, Rogers and Heal, my colleagues and I have disclosed procedures for obtaining from leaves, stems and roots of plants of the genus Ryania, by extraction, an alkaloidal fraction having marked insecticidal activity. The

procedures there disclosed, however, lead only to extracts or concentrates in solvent media, or dry amorphous powders, which comprise alkaloidal mixtures. Not all of the alkaloidal material in these mixtures is active as an insecticide, and since the composition of these mixtures varies depending on the plant material employed and extraction procedure used, it is difiicult to obtain extracts or concentrates of uniform composition and potency.

I have discovered an improved extraction procedure by which it is possible for the first time to obtain an insecticidally active alkaloid, ryanodine, in pure, crystalline form from woody stem and root materials of plants of the genus Ryania. Ryanodine can be obtained from various Ryania species, but the preferred source is Ryania speciosa Vahl. since this species is most extensively available.

Regarded in certain of its broader aspects the novel process in accordance with the present invention comprises preparing, either directly or indirectly from a chloroform extract, an aqueous solution of Ryania extractives, extracting the aqueous solution with an organic solvent of the class consisting of ether and amyl acetate, evaporating the organic solvent extract to dryness in vacuo forming an amorphous residue, dissolving the residue in a solvent of the class consisting of acetone and ether and crystallizing out crude ryanodine, and purifying the crude material by recrystallization from ether.

Pure ryanodine thus obtained, forms clear, colorless crystals melting consistently at 219-220 C., and has a specific rotation of +26 (0. 1.02 in methanol). It is a neutral substance, soluble in water, alcohol, acetone, ether and chloroform, difiicultly soluble in benzene, and insoluble in petroleum ether. The ultraviolet absorption spectrum shows a maximum at 2685 A.

(Ela

in alcohol solution.

The molecular weight of ryanodine determined by the East method is about 487. Microanalytical data are in agreement with the formulas C25-26H35 37NO9, having molecular Weights of 493 and 508. Functional group analysis indicate that ryanodine contains six or seven active hydrogen atoms, a minimum of three carbon-methyl groups, and no acetyl, methoxy, or nitrogenmethyl groups.

Ryanodine is many times more active insecticidally then Ryania extracts prepared as described in said United States Patent No. 2,400,- 295. In view of the high potency which is consistently obtained in pure ryanodine, it will be evident that ryanodine is a valuable substance for use in the compounding of insecticidal sprays and dusts. It will also be noted that since ryanodine is a colorless substance, it is particularly suited for inclusion in moth-proofing preparations for white and light colored fabrics.

The preparation of ryanodine in accordance with the present invention is carried out by extracting stem and root material of Ryania plants with water or with an organic solvent such as chloroform or methanol. When an organic solvent is used, better extraction is obtained with wet wood than with dry wood. In practice, extraction with water or chloroform is favored since these solvents introduce least complication in succeedingsteps of the process.

When water is used for extraction the plant material is preferably extracted twice with boiling water, approximately one gallon of water per pound of plant material, for three-hour periods. After separation of the plant material, the combined aqueous extract is concentrated to about /30 the original volume in vacuo and clarified by centrifuging. The resulting concentrate is exhaustively extracted with ether or with amyl acetate and the organic solvent extract is concentrated to dryness in vacuo. The light brown fluffy solid thus obtained is dissolved in ether or acetone, the solution is concentrated to about /3 to /2 volum and allowed to stand to separate crystalline ryanodine. Cooling to about 0-5 C. after crystallization begins fosters complete crystallization. The crude material obtained on initial crystallization melts at about 142-150 C., and this is refined to obtain pure ryanodine, melting at 219-220 C. by five or six recrystallization from ether.

When chloroform is used for extraction a quantity of Ryania plant materials, preferably ground to approximately 20-mesh, is thoroughly wetted with water, and placed in a Soxhlet type extraction apparatus, and extracted with chloroform but it is to be understood that these examples are given by way of illustration and not of limitation.

Example I One kilogram of root of Ryam'a speciosa Vahl., ground to approximately 20-mesh, was extracted twice by stirring with 8-liter volumes of boiling water for three-hour periods. The plant material was removed after the extractions by means of a basket centrifuge. The combined extracts were concentrated in vacuo to a volume of 500 ml. The concentrate was clarified by centrifuging and then extracted continuously with ether for sixteen hours. The filtered ether extract was concentrated to dryness in vacuo, final traces of water being removed by an oil pump. The residue, 2. light brown, fluffy solid, weighed 4.1 g. It was redissolved in 250 ml. of ether by refluxing for a short time. The ether solution was concentrated to 100 ml.-volume, then allowed to stand at room temperature until crystallization began (one to two days). The solution was then placed in an icebox to complete the crystallization. (Premature chilling caused precipitation of amorphous material which hindered crystallization.) The yield of crude material which melted at 142-150 was 1.150 g. By six recrystallizations of the crude material from ether, 180 mg. of pure ryanodine which melted constantly at 2l9220 was obtained: a 25 26 (0. 1.02 in methyl alcohol). All samples were dried to constant weight at 100 in vacuo before analysis.

Anal. Calcd. for C25H35NO9Z C, 60.83; H, 7.15; N, 2.84; M. W. 493.5; 7 active H, 1.43; 3(C)CH3, 9.14. Calcd. for CzsHa'zNOet C, 61.52;H, 7.35; N, 2.76; M. W. 507.57. Found C, 61.01, 60.89, 60.78, 61.29, 61.37, 61.22, 61.26 (average 61.14); H, 7.29, 7.41, 7.51, 7.45, 7.26, 7.00, 7.10 (average 7.29); N, 2.74, 2.83, 2.82, 2.79, 2.94 (average 2.82); M. W. (Rast), 486,6; active H (hot) 1.21, 1.44, 1.42, 1.18 (average 1.31); (C)CH3, 9.10. Methoxy, N'- methyl and acetyl determinations were negative.

Example II Five hundred grams of stem of Ryania speciosa Vahl. ground to approximately 20-mesh, was thoroughly wetted by admixture with 800 ml. of water. The wet wood was placed in a Soxhlet apparatus and extracted with chloroform for twenty four hours. The chloroform extract was concentrated to. a volume of 750 mil., then extracted with four 300-ml. volumes of water. The combined aqueous extracts were extracted with four 20,0-ml. volumes of amyl acetate. The amyl acetate extract was concentrated to dryness in vacuo, giving 1.925 g. of a light brown powder. Crystallization of this material from concentrated acetone solution yielded 0.893 g. of crude alkaloid melting at 155-160. Pure ryanodine was obtained in 16% yield after five recrystallizations from ether.

The following examples are given to show the comparative insecticidal and mothproofing properties of ryanodine and Ryania extracts of the type disclosed in said U. S. Patent No. 2,400,295.

Example III Adults of the German cockroach, Blatella germamca, were anaesthetized with carbon dioxide, and small measured quantities of solutions of ryanodine and of total aqueous Ryania extractives or principles were deposited on their ventral thoracic surfaces. The dosages of insecticide were varied by adjusting the concentration of the solutions. Dosage-mortality curves were established for ryanodine and for crude aqueous extractives of Ryam'a speciosa. stem by this method using a test of four days duration.

The L. D. 50' established by this test for these materials follows Ryanodine- Femaleapprox. 3.5 mgJkg. of body weight Male-approx. 1.75 mg./kg. of body weight Ryania aqueous extractives 7R3344- Female-approx. 250 mg. extvs./kg. of body wt. Maleapprox. mg. extvs./kg. of body wt.

The result of this test show ryanodine to be approximately 70 times as potent as the aqueous extractives.

' Example IV Samples of woolen fabric were impregnated with aqueous solutions of the crude aqueous extractives of Ryam'a. speciosa, stem and with alcoholic solutions of ryanodine to give varying concentrations of the materials on the fabric. The treated fabrics were then permitted to dry. These samples of the treated fabrics were subjected to the feedingattack of larvae of the web- Ryanodine-0.001% of weight of wool Ryania aqueous extractives--0.056% of weight of wool Various changes and. modifications can be made in the procedures herein described without departing from the spirit and scope of the present invention, and I am to be limited only by the appended claims.

I claim:

' 1. The process that comprises extracting an aqueous solution of Ryania extractives with ether, evaporating the ether extract to dryness in vacuo forming an amorphous residue, dissolving the residue in a solvent of the class consisting of acetone and ether and crystallizing out crude ryanodine, and purifying the crude ryanodine by recrystallization from ether until the crystalline product obtained has a consistent melting point of 219-220 C.

2. The process that comprises extracting an aqueous solution of Ryania extractives with ether, evaporating the ether extract to dryness in vacuo forming an emorphous residue, dissolving the residue in ether and crystallizing out crude ryanodine, and purifying the crude ryanodine by recrystallization from ether until the crystalline product obtained has a consistent melting point of 219-220 C.

3. The process that comprises extracting Ryania plant material With water by boiling in about one gallon of water per pound of Ryania plant material for about three hours, removing the residual plant material and concentrating the aqueous extract to about 1/30 the original volume, extracting the concentrated aqueous solution with ether, evaporating the etherextract to dryness in vacuo forming an amorphous residue, dissolving the residue in a solvent of the class consisting of acetone and ether and crystallizing out crude ryanodine, and purifying the crude ryanodine by recrystallization from ether until the crystalline product obtained has a con-- sistent melting point of 219-220 C.

4. The process that comprises extracting ground stems and roots of Ryamia speciosa Vahl. with water by boiling in about one gallon of water per pound of Ryania plant material for about three hours, removing the residual plant material 20 2,400,295

and concentrating the aqueous extract to about 1/30 the original volume, extracting the concentrated aqueous solution with ether, evaporating the ether extract to dryness in vacuo forming an amorphous residue, dissolving the residue in a solvent of the class consisting of acetone and ether and crystallizing out crude ryanodine, and purifying the crude ryanodine by recrystallization from ether until the crystalline product obtained has a consistent melting point of 219- 220 C.

EDWARD F. ROGERS.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Name Date Folkers et al May 14, 1946 Number 

1. THE PROCESS THAT COMPRISES EXTRACTING IN AQUEOUS SOLUTION OF RYANIA EXTRACTIVES WITH ETHER, EVAPORATING THE ETHER EXTRACT TO DRYNESS IN VACUO FORMING AN AMORPHOUS RESIDUE, DISSOLVING THE RESIDUE IN A SOLVENT OF THE CLASS CONSISTING OF ACETONE AND ETHER AND CRYSTALIZING OUT CRUDE RYANODINE, AND PURIFYING THE CRUDE RYANODINE BY RECRYSTALLIZATION FROM ETHER UNTIL THE CRYSTALLINE PRODUCT OBTAINED HAS A CONSISTENT MELTING POINT OF 219-220* C. 